M. hyopneumoniae is the primary pathogen of enzootic pneumonia, an economically important and globally, highly prevalent disease in pigs. M. hyopneumoniae is also considered to be one of the primary agents involved in the porcine respiratory disease complex (PRDC; see E. L. Thacker; 2006; Diseases of Swine; 9th edition; Blackwell publishing; editor: B. E. straw et al.; pages 701-717). Mycoplasmal pneumonia is most commonly spread through direct contact with respiratory tract secretions of infected swine, but may also be spread through airborne transmission. Clinically, mycoplasmal pneumonia can be described as a chronic disease with high morbidity and low mortality. The principal sign is a chronic nonproductive cough, with inappetence, labored breathing (thumping), and unthriftiness, or an unhealthy appearance.
Despite the low mortality of enzootic pneumonia due to infection with M. hyopneumoniae, this disease is a significant economic problem for swine producers worldwide. An approximate herd average lung lesion score of 10 depresses growth rate by 5% (37.5 g/day) (see Burch; 2007; Cost of Disease—Enzootic Pneumonia; Pig World, February 2007). Additionally, a herd average lung lesion score of 10 reduces the Feed Conversion Rate by about 4.5% (see Straw B. E., et al.; 1986; Examination of Swine at Slaughter. Part II. Findings at Slaughter and their Significance). Consequently, prevention of this disease would provide significant commercial benefit to swine husbandry (see e.g. Maes et al.; 1999; Vaccine, vol. 17, pages 1024-1034).
At present, commercial vaccines to protect healthy swine against the clinical signs caused by M. hyopneumoniae are the major tools for reducing the clinical impact of the disease. Most commercial vaccines consist of adjuvanted whole cell preparations. Commercial vaccines differ significantly in respect to their administration regime. International application WO 92/03157 A1, for example, discloses a vaccine against infections by M. hyopneumoniae that comprises a binary ethyleneimine (BEI) inactivated M. hyopneumoniae strain and 0.2% (w/v) (i.e. 2 mg/ml) acrylic acid.
Over the past few years single-dose vaccines have been developed which allow a “one shot” immunization to reach an acceptable level of immunity against the respective infection. Additionally, technical progress has been made with respect to the apparatus used in the vaccination step itself. For example, US 2008/0014221 A1 discloses an immunization method against M. hyopneumoniae infections that administers a single dose vaccine with a liquid, jet, needle-free injector.
An example for the development of single-dose vaccines can be seen in the publication “Studies of the field efficacy and safety of a single-dose Mycoplasma hyopneumoniae vaccine for pigs” (2002) by A. Dawson, et al., Veterinary Record, vol. 151, pages 535-538. It describes a field study of three- to five-week-old pigs, immunized once by a whole cell vaccine of M. hyopneumoniae comprising the adjuvant AMPHIGEN® adjuvant from Pfizer (New York, USA), in comparison to a control group being immunized with physiological saline solution. According to the manufacturer's information (see “Next generation adjuvant system: Key to enhanced protection conferred by BVDV (Types 1 and 2) components of CATTLEMASTER® GOLD™” adjuvant; Pfizer Animal Health, Technical Bulletin, July 2004, page 4), the AMPHIGEN® adjuvant is a composition of a lecithin-derived phospholipid and a glycolipid surfactant in a light, highly refined oil.
The disadvantages of an oily adjuvant (e.g. adverse local reactions such as lumps, abscesses, and granulomas at the injection site) are overcome in AMPHIGEN® adjuvant by the addition of lecithin, which “naturalizes” the oil and makes it more accessible to the cells of the immune system. Aluminum hydroxide, a commonly used adjuvant in vaccines, and conventional oil adjuvants lack this compatibility. Additionally, because of its low viscosity, AMPHIGEN® adjuvant makes the respective vaccine highly syringeable and minimizes reactions upon injection. In comparison, water-in-oil based vaccines are, due to their high viscosity, extremely difficult to draw up into a syringe and are additionally often associated with long-lasting reactions at the site where the products are administered. In the two described Dawson studies, the volume of the vaccine was 2 ml, comprising the antigen and the adjuvant or the saline, respectively. As a result, the vaccinated animals had significantly lower lung lesion scores than the control animals (P=0.0022 and in a parallel group P=0.0056).
A further development, produced by the same manufacturer, available under the trade name RESPISURE-ONE® adjuvant (see pfizerah.com/product_overview; printed Apr. 10, 2008) also comprises a whole cell bacterin of M. hyopneumoniae and the oil-in-water adjuvant AMPHIGEN®. This bacterin is described as able to elicit 25 weeks of immunization after administering a single dose and as being effective in pigs one week of age or older. But, the total volume of the applied dose of 2 ml is still not optimal. Smaller volumes would be advantageous. Further, the viscosity of this vaccine—though ameliorated in comparison to other oil comprising adjuvants—still needs to be improved.
Another route to enhance the immunogenicity of an antigen by the admixture of an adjuvant preparation has been chosen by Wyeth/Fort Dodge Animal Health (FDAH; Madison, N.J., USA). Fort Dodge markets a M. hyopneumoniae bacterin under the trade name SUVAXYN RESPIFEND® MH for use as a vaccine. This vaccine contains 2 mg/ml CARBOPOL® carbomer as an adjuvant and is recommended as a two-dose vaccine for pigs at least one-week old, with the second dose to be administered two to three weeks after the first vaccination. But, a two-dose vaccine has the obvious disadvantage of requiring a second handling of the animals in order to provide full protection against disease.
A further development designed for a single administration is disclosed in the international application WO 02/49666 A2 that is based on U.S. application 60/256,637. The disclosed M. hyopneumoniae vaccine comprises an adjuvant mixture comprising an acrylic acid polymer and a mixture of a metabolizable oil and a polyoxyethylene-polypropylene block copolymer. This special admixture of a certain oil with the mentioned other ingredients is described as enhancing the immunogenicity of the bacterin so as to elicit protective immunity after a single dose of the vaccine. It is explicitly disclosed that the acrylic acid polymer is a carboxymethylene polymer, esp. CARBOPOL® 934P (Carbamer 934P) carbomer which may be present in the amount of about 2 ml/l. Such acrylic acid polymers were formerly marketed by B.F. Goodrich, now Noveon, Inc. (Pedricktown, N.J., USA) as CARBOPOL® 934 P NF and 941 NF carbomers. They are polymers of acrylic acid cross-linked with polyallylsucrose and have the chemical formula (CH2CHOOOH)n. These polymers form aqueous gels which suitably formulate with aqueous carriers. In explicitly disclosed modes the metabolizable oil is squalene or squalane, and the polyoxyethylene-polypropylene block copolymers are the nonionic surfactants marketed by BASF (Ludwigshafen, Germany) as PLURONIC® E L121, L61, L, 81 or L101 nonionic surfactants. Such vaccines elicit four months duration of immunity (DoI) in pigs induced by one-dose vaccination of the respective vaccine at an age of three weeks. These vaccines comprise a significant portion of oil and are applied at total volumes of 2 ml.
There remains a need to develop a vaccine against the swine pathogen M. hyopneumoniae that reduces the clinical signs of enzootic pneumonia and allows a long term effective immunity after a single administration of the vaccine, combined with an early onset of effective immunity. The design of the composition should avoid the negative side effects that accompanied oil adjuvanted vaccines, especially an undesirable high viscosity and local reactions at the injection site. Such a vaccine should additionally have the capacity to be effectively applied in doses less than 2 ml, preferably not more than 1 ml, to elicit effective immunity.